1. Field of the Invention
This invention relates to immunology, and, more specifically, to the preparation and use of immunopotentiating agents which are capable of eliciting, enhancing and/or otherwise modifying immune responses. These agents, through their ability to elicit or enhance cellular or humoral responses, have potential utility in a variety of disease conditions wherein immunotherapy might be expected to provide a benefit.
2. Description of Related Art
The body""s immune system serves as a defense against a variety of conditions, including, e.g., injury, infection and neoplasia, and is mediated by two separate but interrelated systems, the cellular and humoral immune systems. Generally speaking, the humoral system is mediated by soluble products, termed antibodies or immunoglobulins, which have the ability to combine with and neutralize products recognized by the system as being foreign to the body. In contrast, the cellular immune system involves the mobilization of certain cells, termed T-cells, that serve a variety of therapeutic roles.
(a) The Immune System
The immune system of both humans and animals include two principal classes of lymphocytes: the thymus derived cells (T cells), and the bone marrow derived cells (B cells). Mature T cells emerge from the thymus and circulate between the tissues, lymphatics, and the bloodstream. T cells exhibit immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection). T cells act against or in response to a variety of foreign structures (antigens). In many instances these foreign antigens are expressed on host cells as a result of infection. However, foreign antigens can also come from the host having been altered by neoplasia or infection. Although T cells do not themselves secrete antibodies, they are usually required for antibody secretion by the second class of lymphocytes, B cells.
There are various subsets of T cells, which are generally defined by antigenic determinants found on their cell surfaces, as well as functional activity and foreign antigen recognition. Some subsets of T cells, such as CD8+ cells, are killer/suppressor cells that play a regulating function in the immune system, while others, such as CD4+ cells, serve to promote inflammatory and humoral responses. (CD refers to cell differentiation cluster; the accompanying numbers are provided in accordance with terminology set forth by the International Workshops on Leukocyte Differentiation (5). A general reference for all aspects of the immune system may be found in (1)).
(b) T Cell Activation
Human peripheral T lymphocytes can be stimulated to undergo mitosis by a variety of agents including foreign antigens, monoclonal antibodies and lectins such as phytohemayglutinin and concanavalin A. Although activation presumably occurs by binding of the mitogens to specific sites on cell membranes, the nature of these receptors, and their mechanism of activation, is not completely elucidated. Induction of proliferation is only one indication of T cell activation. Other indications of activation, defined as alterations in the basal or resting state of the cell, include increased lymphokine production and cytotoxic cell activity.
T cell activation is an unexpectedly complex phenomenon that depends on the participation of a variety of cell surface molecules expressed on the responding T cell population (2,3). For example, the antigen-specific T cell receptor (TcR) is composed of a disulfide-linked heterodimer, containing two clonally distributed, integral membrane glycoprotein chains, xcex1 and xcex2, or xcex3 and xcex4, non-covalently associated with a complex of low molecular weight invariant proteins, commonly designated as CD3 (the older terminology is T3) (2,4).
The TcR xcex1 and xcex2 chains determine antigen specificities (6). The CD3 structures are thought to represent accessory molecules that may be the transducing elements of activation signals initiated upon binding of the TcR xcex1xcex2 to its ligand. There are both constant regions of the glycoprotein chains of TcR, and variable regions (polymorphisms). Polymorphic TcR variable regions define subsets of T cells, with distinct specificities. Unlike antibodies which recognize soluble whole foreign proteins as antigen, the TcR complex interacts with small peptidic antigen presented in the context of major histocompatibility complex (MHC) proteins. The MHC proteins represent another highly polymorphic set of molecules randomly dispersed throughout the species. Thus, activation usually requires the tripartite interaction of the TcR and foreign peptidic antigen bound to the major MHC proteins.
With regard to foreign antigen recognition by T cells the number of peptides that are present in sufficient quantities to bind both the polymorphic MHC and be recognized by a given T cell receptor, thus inducing immune response as a practical mechanism, is small. One of the major problems in clinical immunology is that the polymorphic antigens of the MHC impose severe restrictions on triggering an immune response. Another problem is that doses of an invading antigen may be too low to trigger an immune response. By the time the antigenic level rises, it may be too late for the immune system to save the organism.
The tremendous heterogeneity of the MHC proteins among individuals remains the most serious limiting factor in the clinical application of allograft transplantation. The ability to find two individuals whose MHC is identical is extremely rare. Thus, T cells from transplant recipients invariably recognize the donor organ as foreign. Attempts to suppress the alloreactivity by drugs or irradiation has resulted in severe side effects that limit their usefulness. Therefore, more recent experimental and clinical studies have involved the use of antibody therapy to alter immune function in vivo. The first successful attempt to develop a more selective immunosuppressive therapy in man was the use of polyclonal heterologous anti-lymphocyte antisera (ATG) (7, 8, 9).
Clinical trials of the ATG treatment suggested a significant reduction of early rejection episodes, improved long term survival and, most importantly, reversal of ongoing rejection episodes. However, the results were often inconsistent due to the inability to standardize individual preparations of antisera. In addition, the precise nature of the target antigens recognized by the polyclonal reagents could not be defined, thus making scientific analysis difficult. The advent of monoclonal antibody (mAb) technology provided the basis for developing potentially therapeutic reagents that react with specific cell surface antigens which are involved in T cell activation.
(c) Effect of Monoclonal Antibodies on the Immune System
Monoclonal antibodies (mAb) were developed by Kohler and Milstein in 1975. The methods generally used to produce mAb consist of fusing (hybridizing) two types of somatic cells: (1) a neoplastic myeloma cell line; and (2) a normal B lymphocyte obtained from an immunized animal. The result is called a hybridoma which is characterized by immortal growth and the ability to secrete antibodies specific for the immunization antigen.
One of the clinically successful uses of monoclonal antibodies is to suppress the immune system, thus enhancing the efficacy of organ or tissue transplantation. U.S. Pat. No. 4,658,019, describes a novel hybridoma (designated OKT3) which is capable of producing a monoclonal antibody against an antigen found on essentially all normal human peripheral T cells. This antibody is said to be monospecific for a single determinant on these T cells, and does not react with other normal peripheral blood lymphoid cells. The OKT3 mAb described in this patent is currently employed to prevent renal transplant rejection (10).
One unexpected side effect of the OKT3 therapy was the profound mitogenic effect of the mAb in vivo (28). Although anti-CD3 mAb has been shown to activate T cells in vitro to produce various lymphokines, etc. (11), OKT3 has not been previously used to stimulate the immune system in vivo.
In addition, other cell surface molecules have been identified that can activate T cell function, but are not necessarily part of the T cell surface receptor complex.
Monoclonal antibodies against Thy-1, TAP, Ly-6, CD2, or CD28 molecules can activate T cells in the absence of foreign antigen in vitro (12, 13,14,15,16). Moreover, certain bacterial proteins although differing in structure from mAbs, also have been shown to bind to subsets of T cells and activate them in vitro (17). Although some of these agents, in vitro effects have previously been demonstrated, in vitro activity is often not a reliable predictor of in vivo effects.
(d) Immune System and Tumor Growth
One cause of malignant tumor growth is believed to be the inability of the immune system to respond appropriately to tumor antigen. For example, malignant progressor tumors are only weakly immunogenic and can evade host recognition and rejection. Both specific and non-specific effector pathways have been implicated in tumor immunity. Treatment by immunotherapy is aimed at remedying defects in the immune weaponry. The aim of immunotherapy has been the enhancement of one or both of these pathways. One potential approach to therapy is to activate host antitumor cellular effector mechanisms.
Historically, non-specific adjuvants such as BCG or pertussis have been used to augment immune responses. In normal individuals these adjuvants amplify immune responses by providing non-specific stimuli that enhance overall immunity. However, these adjuvants do not selectively act on T cells, or subsets of T cells, and have not been shown capable of overcoming immunodeficiency states. Unfortunately, current modes of immunotherapy which induce non-specific effector cells are not effective enough in potentiating anti-tumor responses (18). Recently, immunotherapy regimens which utilize the ability of the immune system to recognize tumor antigens in a specific manner, for instance utilizing specific tumor-infiltrating lymphocytes, for immunotherapy, have been suggested to result in superior anti-tumor immunity (19). Thus, current efforts toward developing more efficacious forms of immunotherapy have focussed on specific anti-tumor response and memory-following antigen recognition. One approach that has not previously been accomplished has been the in vivo administration of T cell activating mAbs to promote anti-tumor activity.
(e) Defenses Against Viral Infections
Human immunodeficiency virus (HIV), the biologic agent of AIDS, causes a persistent infection associated with profound immunosuppression resulting in susceptibility to opportunistic infections. Immunological responses to HIV infection require the development of both humoral and cell mediated effector mechanisms; however current efforts in treatment and vaccine design have fallen short of success either due to the immunodeficiency associated with the viral infection, or to the low immunogenicity of the vaccine (20). The development of a safe and effective vaccine against infection with human immunodeficiency virus (HIV) is complicated by a lack of understanding of protective immunity to HIV and disease development, and the absence of an adequate and convenient animal model for studying HIV infection.
Because HIV can be transmitted as either a cell-free or cell-associated virus, a protective immune response against HIV infection will likely require both humoral and cell-mediated immunity, including neutralizing antibody against HIV, antibodies involved in antibody-dependent cellular cytotoxicity and cytotoxic lymphocytes. All of these activities involve virus-specific T cells. T cell activation requires potent in vivo immune responses to foreign antigens such as viruses.
In individuals infected with HIV, two components of the immune system are suboptimal and, therefore, the ability to generate an immune response in these individuals has been compromised. First, the reduced frequency of antigen-reactive CD4+ T cells is apparently not sufficient to mount an appropriate immune response to HIV, especially if the quantity of HIV antigen is low. CD4 is a membrane protein that acts as a binding site and entry port into CD4+ lymphocytes for the human immunodeficiency virus-type 1 (HIV-1) (21). Second, all immune responses are dependent on the ability of T cells to recognize processed antigen associated with major histocompatibility antigens (MHC). Any vaccine approach which utilizes HIV peptides or inactivated virus antigen must depend on the ability of antigenic peptides to bind the appropriate MHC antigens necessary to initiate an immune response. Given the tremendous polymorphism of the MHC antigens expressed in the population, and the variation of the HIV virus, developing a successful HIV vaccine for general use is difficult and has not yet been successful.
(f) Problems in Developing Vaccines to Weakly Immunogenic Antigens
The usefulness of certain peptides, proteins or other potential or desired immunogens in vaccines can be limited by several critical factors. For example, low immunogenicity of the peptide or other structure which one desires to employ can be a difficult problem to overcome, particularly with smaller peptides and those peptides which do not contain appropriately strong B- and/or T-cell potentiating sequences. Such peptides are typically only weakly immunogenic at best. Moreover, to be of widespread applicability, the peptides chosen must be capable of inducing an immune response in a majority of the population.
It has been difficult to protect against attack by organisms such as the HIV virus or to provide tumor immunity for several reasons. For example, genetic differences exist among individuals at the major histocompatibility locus, which limits the system""s ability to respond to individual small peptides. Thus, the various components of the immune response, including the T cells and B cells, may not interact appropriately in generating a response to non- or weakly-immunogenic small peptides. Attempts to improve peptidyl immunogenicity have centered principally on the use of adjuvants such as alum or complete Freund""s adjuvant. However, prior adjuvants such as these have proven to be inadequate for various reasons, including an inability of the adjuvant to specifically enhance T or B cell activity and the inability of the adjuvant to overcome the severe limitations of MHC restriction.
Although glimpses into the defense mechanisms of the body""s own immune system have been provided by in vitro studies and by observation of some in vivo reactions, there is a serious lack of successful therapeutic methods to augment immunity in vivo. Improved compositions and/or methodology for eliciting or enhancing cellular or humoral responses in mammals are needed both to provide animal models for investigation of therapeutic regimes, to provide novel means of preparing improved immune system-directed products such as improved immunotherapeutic antibodies, and to advance treatment and possible immunization, e.g., for conditions such as HIV, cancer and infections.
The present invention is concerned with a broad array of embodiments, generally involving methods and compositions for potentiating one or more aspects of the immune response of a human or other animal having an immune system, as well as to products which may be derived out of the use of these methods and compositions. Generally speaking, the invention concerns essentially four categories of what may be referred to broadly as immunopotentiating or immunoactivating compositions: 1) individual immunopotentiating agents which are used to potentiate one or more aspects of the immune system; 2) immunopotentiating xe2x80x9cadjuvantxe2x80x9d compositions wherein immunopotentiating agents are employed essentially as xe2x80x9cadjuvantsxe2x80x9d to improve the body""s immune responsiveness to other compounds which are co-administered with, or included in with, compositions containing the immunopotentiating agent; 3) immunopotentiating conjugates wherein the immunopotentiating agent is actually chemically coupled to the compound against which an immune response is desired; and 4) products derived from the administration of one or more of the foregoing, including, e.g., antibodies, antibody-producing cells, T-cells, potentiated bone marrow progenitor cells, and the like.
The term xe2x80x9cactivationxe2x80x9d is generally defined to refer to any change induced in the basal or resting state of T or B cells. This includes, but is not limited to, increased cell proliferation and DNA synthesis, lymphokine and cytotoxic cell production, a rapid rise in intracellular calcium, release of water soluble inositol phosphates, increased IL-2 receptor expression, enhanced proliferative response to IL-2, and enhanced responses to foreign antigens or MHC (23). In contrast, the term xe2x80x9cimmunopotentiatingxe2x80x9d is classically defined as the ability to produce an effect on the immune system which enhances the system""s ability to respond to foreign antigens. Thus, immunopotentiation may affect the cellular response, humoral response, or both. Exemplary indices of immunopotentiation include, but are not limited to, cell proliferation, increased DNA synthesis, increased production of lymphokines, increased production of cytotoxic cells, calcium efflux, or any other change that raises the cell above the basal or resting state.
While one can consider there to be a distinction between the terms xe2x80x9cpotentiationxe2x80x9d and xe2x80x9cactivationxe2x80x9d, in the context of the present invention the use of the term xe2x80x9cpotentiationxe2x80x9d is intended to include both potentiation and activation. Thus, the immunopotentiation achieved by the methods and agents of this invention may affect all T cells, certain subsets of T cells, or B cells, depending on the nature of the agent(s) and their dose levels. One of the objects of this invention is to provide means for fine tuning immunopotentiation, allowing one to target T and/or B cell response depending on the nature of the clinical condition to be treated.
Accordingly, in certain general embodiments, the present invention concerns the preparation and use of immunopotentiation agents, whether used alone as a direct immunopotentiation agent, or combined with other compounds, either covalently or simply admixed in the same composition. In the context of the present invention, the term immunpotentiation agent is intended to include immunopotentiating antibodies, as well as certain bacterial proteins which have been determined to have profound immunopotentiation actions.
In terms of antibodies, useful immunopotentiation agents will generally involve antibodies against a cell surface epitope of T-cells wherein binding of the antibody to the surface epitope of the T-cell will result in immunopotentiation. An exemplary antibody is anti-CD3 (e.g., OKT3), previously known only to be immunosuppressive and not previously known to be immunopotentiating. The present inventor has surprisingly discovered that, in fact, when anti-CD3 is administered at relatively low doses (e.g., on the order of 100 to 200 xcexcg/kgm body weight), rather than being immunosuppressive it exhibits a very profound immunopotentiation effect. The reason for this appears to include but may not be limited to the induction of lymphokines, the proliferation of T cells, or even the progression of T cells from a naive to memory state.
While anti-CD3 is a useful immunopotentiation agent, numerous other immunopotentiation antibodies are contemplated to be within the scope of the present invention. Such antibodies are defined generally as antibodies which recognize and activate a T cell activation molecule or epitope on the cell surface of T cells. For example, monoclonal antibodies which are particularly useful in the practice of the present invention will comprise those directed against the T cell variable or constant epitopes on the cell surface of T cells. The T cell activation molecules which are expressed on the cell surface may be either those associated with the T cell receptor complex or those with the antigens distinct from, that is not physically associated with, TcR on the cell surface. Specific embodiments of T cell activation molecules comprise either the variable or the constant region epitopes as expressed on the antigen specific T cell receptor polymorphic chains, e.g., xcex1, xcex2, xcex3, and xcex4 chains.
Embodiments of the non-polymorphic TcR associated CD3 chains against which monoclonal antibodies may be directed for use as immunopotentiating agents are the xcex3, xcex4, xcex5 or xcex6 chains. Monoclonal antibodies have been developed against some of these chains, as exemplified by OKT3, SP34, UCHTI or 64.1 (68-70). Among the T cell surface antigens which are distinct from, and not physically associated with, TcR, are CD2, CD28, Thy-1, and the activation epitopes expressed on members of the Ly-6 protein family.
As noted, the immunopotentiation agents of the present invention will also include certain potentiating bacterial proteins such as bacterial enterotoxins, exemplified by staphylococcal enterotoxin B (SEB). SEB is now known to activate T-cells and provide surprisingly profound and subset-specific potentiation. As with some of the potentiating antibodies, the mechanism of how enterotoxins function to stimulate the immune system is not entirely clear, but could involve lymphokine production or T cell proliferation. While SEB comprises a preferred enterotoxin for immunopotentiation purposes, the invention contemplates that other similar enterotoxins, such as staphylococcal enterotoxins A, C1, C2, D, E, toxic shock syndrome toxin (TSST), exfoliating toxin (ExFT) and likely even mycoplasma arthriditis substance, will find similar utility.
For use directly as immunopotentiation agents, one will generally desire to first combine the agent in a pharmacologically suitable vehicle, such as combining with an appropriate diluent or buffer, in an amount and concentration that is appropriate to effectuate potentiation of the immune system when administered. Typically, in the case of, e.g., anti-CD3, one will desire to provide parenteral compositions having from about 0.1 mg/ml to about 1 mg/ml, in order to allow the parenteral administration of appropriate amounts of the antibody.
As mentioned, the present invention contemplates the use of these immunopotentiating agents in immunogen containing compositions such as vaccines, where the agents serve as xe2x80x9cadjuvantsxe2x80x9d to improve the immunogenicity of other components of the composition. Thus, it is contemplated that through the use such agents as xe2x80x9cadjuvantsxe2x80x9d, the preparation of useful vaccines using only weakly or non-immunogenic molecules not previously known to function as immunogens is enabled. In such embodiments, the immunopotentiating protein is admixed or otherwise coadministered with the molecule against which an immune response is desired, with the immunopotentiating agent being present in amounts effective to promote potentiation upon administration to the particular subject.
It is proposed that still further advantages will be realized where the immunopotentiation agent is actually conjugated to the molecule against which an immune response is desired. These conjugates are referred to in the context of the present invention as xe2x80x9cheteroconjugatesxe2x80x9d. As used herein, an immunopotentiating heteroconjugate is defined as an immunopotentiating agent conjugated to a second protein or other molecule against which an immune response is desired, the conjugation being in the nature of a chemical or molecular crosslink.
Exemplary embodiments of the second molecule include proteins which comprise amino acid sequences or other potential determinants (e.g., a non-amino acid determinant such as a sialo group) against which a cellular or humoral immune response is desired. However, as with the adjuvant embodiments, the present invention also contemplates the use of non-protein molecules, such as glycolipids, carbohydrates or even lectins, as the second molecule against which an immune reaction is sought. All that is required for use in the context of heteroconjugates is that one be capable of conjugating the molecule to the immunopotentiation agent.
The immunopotentiating protein of the heteroconjugate may also be formed by linking two monoclonal antibodies directed against two distinct but specific T cell epitopes. A specific embodiment of this type of immunopotentiating protein is anti-CD3 coupled with anti-CD4 to form a CD4 subset-specific immunopotentiating protein. It has been demonstrated that administering of such a bispecific antibody construct in vivo activates T cells to a surprising degree (see, e.g., U.S. Pat. No. 4,676,980 to Segal, and FIG. 14 herein). It is proposed by the present inventor that one may employ such a bispecific antibody construct as the immunopotentiating ligand of heteroconjugates formed with a second protein against which an immune response is desired.
Use of the heteroconjugate disclosed in this invention is believed to aid in directing the immune attack to specific epitopes, via the attached compound or protein. Thus, it is proposed that a particular utility of these embodiments will be found in the treatment of diseases such as cancer, where only weakly immunogenic tumor-specific or tumor-associated proteins are found to appear on the cell surface of many tumors, or are found to characterize the tumor.
It is proposed that other particularly important applications are in the context of viral-specific or viral-associated epitopes, particularly those where the immune system may be compromised, such as in the case of HIV infections. Moreover, amino acid sequences homologous to those derived from genes in a bacteria, fungus, protozoal or metazoal parasite, may also be used as conjugating agents.
Of course, where desired, the epitopes used as second proteins may either be derived from the cell surface or extracted from cells which are infected or diseased. Alternatively, amino acid sequences can be made synthetically by standard chemical synthetic or molecular biology methods. Thus, for example, in the case of HIV immunotherapy, amino acid sequences are known for various peptides isolated from the HIV virus gp120 cell membrane which can be employed as embodiments of the second compound, whether it be conjugated to the immunopotentiating protein or simply admixed in an appropriate pharmaceutical vehicle. Preferred embodiments of peptides which may be used as second proteins in a heteroconjugate used to augment immunity against HIV, comprise peptides 18, T1, T2, or peptides derived from the CD4 binding site.
In similar fashion, epitopes specific for amino acid sequences homologous with those expressed on the surface of human hepatitis virus, or extracted from cells infected with the virus may be used as the second protein. Viral specific epitopes may also comprise amino sequences homologous with those expressed on the surface of infected cells.
Immunogenic compositions may be prepared for administration to subjects by including any of the foregoing immunopotentiating compositions, and formulated to include an effective amount of 1) an immunopotentiating protein (i.e., where one desires simply generalized immunopotentiation rather than potentiation that is directed against a second compound), 2) an immunopotentiation protein in combination with a second compound, or 3) an immunopotentiation protein conjugated to such a second compound. The materials that are formulated are preferably rendered pharmacologically acceptable by manufacturing in accordance with good manufacturing practices, extensively dialyzed to remove undesirable small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.
Where such compositions are intended for human administration, one will typically desire to include an amount of immunopotentiation agent that will result in T cell activation in the absence of concomitant immunosuppression. The determination of exact amounts will depend on the particular circumstances, such as the particular condition to be treated, the physical condition of the patient, the type of immunogenic composition that is to be administered, and the like. Thus, e.g., based on murine studies performed by the inventor one can extrapolate in the case of embodiments incorporating immunopotentiating antibodies such as anti-CD3, anti-CD28 or anti-CD2, that suitable formulations should typically include from about 10 ug to about 1000 ug bolus/patient every 14 days or so, and more preferably 100 ug to about 400 ug of the antibody per patient. Similarly, one can extrapolate in the case of enterotoxins that one should typically desire to employ from about 100 ug to about 10 mg of the enterotoxin per dosage, and more preferably about 1 to about 5 mg/dose.
For the preparation of compositions suitable for parenteral administration, the immunogens of the invention may be formulated in oils such as sesame or peanut oil, aqueous propylene glycol, in liposomes or in sterile aqueous solutions. Such solutions are typically suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Additionally, stabilizers in the form of, for example, sorbitol or stabilized gelatin may be included. These particular aqueous solutions may be particularly well suited for intramuscular and subcutaneous injections, as may be preferred for vaccination using antigenic preparations.
The proteins may be formulated into the composition as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isoprophylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual""s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimens for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
Various methods of achieving additional adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70xc2x0 to 101xc2x0 C. for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacterial, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
While administration of the foregoing immunopotentiating compositions will likely find their greatest utility and application in the treatment of human disease, the invention is by no means limited to human application, and is intended to apply to any mammal having an immune system, including, e.g., rodents such as mice, hamsters and rats, primates, rabbits, even farm animals such as cows, pigs, etc. Thus, the invention contemplates, e.g., that certain of the foregoing embodiments will have general applicability wherever one desires to obtain an enhanced immune response against a desired molecule, such as in the initial immunization of animals for hybridoma or even polyclonal antibody development.
While it is contemplated that the nature of the second molecule is not crucial to the successful practice of the invention, it is recognized that the invention will find its greatest utility where the second molecule is a peptide, in that peptides are often notoriously difficult to obtain an immune response against. Thus, it is believed that particular benefits will be realized through the use of peptides having from about 8 to about 100 amino acids in length, and even more preferably, about 8 to about 50 amino acids in length.
In the context of heteroconjugates, it is contemplated that numerous methods for conjugation may be applied, including but not limited to: 1) the formation of biotinavidin bridges; 2) the use of cross linkers such as SPDP to link the functional units; 3) cross-linking of maleimide and SH groups; as well as numerous other possibilities. In general, all that is required is that the cross-linking maintain the integrity of the peptidic antigen and leave unaltered the activating property of the immunopotentiating reagent.
As mentioned above, the present invention contemplates that various useful biological products may be derived through the application of the foregoing immunopotentiating compositions. For example, the adjuvant and heteroconjugate embodiments will provide extremely useful means for preparing antibodies, including monoclonal antibodies. Moreover, it has been found that immunopotentiating antibodies such as anti-CD3 can serve to promote the recruitment of hematopoietic progenitor cells, presumably by stimulating the release of cytokines and lymphokines from activated T-cells. This lends the possibility that such embodiments may be employed to prepare highly active bone marrow for transplantation, or even for administration to bone marrow transplant recipients or those with depleted bone marrow cells to provide a metabolic boost to the marrow. Moreover, it is contemplated that activated T cells themselves will find some utility, such as in anti-tumor therapy that employs tumor-infiltrating lymphocytes.